Several species of mycobacteria, such as M. tuberculosis, M. leprae and M. avium, are intracellular pathogens which survive in the phagosomal compartments of macrophages. Among the salient features of these gram positive organisms are the complex lipid content and the extremely low permeability of their cell walls. The strong immunogenic response to partially purified mycobacterial cell wall proteins has been extensively studied. Nevertheless, little is known about the structure or function of specific proteins embedded in the membrane or associated with the cell wall. I propose to study one class of membrane proteins and their activities - membrane transport proteins- in M. smegmatis, a fast growing non-pathogenic species of mycobacteria which is amenable to genetic and biochemical manipulation. I will focus on sugar (glucose and glycerol) and amino acid (arginine) transport, by isolating and characterizing mutants defective in transport of these nutrients, and cloning the genes regulating transport of three specific nutrients. Glucose is a major carbon and energy source for mycobacteria, and glycerol is a precursor of surface lipids some of which are principle virulence factors in pathogenic species of mycobacteria. In E. coli and other bacteria, arginine nine is transported by a multicomponent system with binding proteins that are members of the ATP binding cassette superfamily of transport proteins; proteins belonging to this family have not been extensively characterized in mycobacteria. In addition, in the macrophage, a primary target cell of mycobacterial infection, arginine is directly required in the production of nitric oxide and other reactive nitrogen intermediates which exhibit marked antimycobacterial effects. These studies will lead to increased understanding of mycobacterial surface proteins, the organization of transport proteins in the cytoplasmic membrane, and the regulation of uptake of nutrients, drugs, and other substances. Among the genetic and molecular biological approaches I will develop are techniques novel for use with mycobacteria. These methods will be generally applicable to important medical problems involving mycobacterial physiology and genetics, such as drug uptake and metabolism, recombinant vaccine development, and the understanding of pathogenic mechanisms.